The protein actin is a major cytoskeletal component of all eukaryotic cells, serving both structural and motility-related roles. In the cell, assembly and disassembly of actin microfilaments are temporally and spatially regulated. This project is aimed at understanding the mechanism of G-actin assembly into F-actin, the role of the hydrolysis of actin-bound ATP, and the cellular control mechanisms for actin assembly. This past year we have shown that monomer-polymer subunit exchange in steady-state F-actin solutions occurs by a diffusion mechanism and not by opposite-end assembly-disassembly (treadmilling). We have also discovered that F-actin alone possesses ATPase activity independent of monomer-polymer interaction; exchange of F-actin-bound ADP for ATP in solution can occur directly by a "breathing" of the polymer structure. The assembly of F-actin with bound ADP has been studied. Even though no nucleotide hydrolysis occurs, the kinetics of assembly are consistent with a rate-limiting nucleation step followed by elongation. We have begun to measure absolute rate constant for the assembly reaction by using a chemically cross-linked actin trimer to bypass the nucleation step. A study has begun to determine the mechanism for control of actin filament assembly in human platelets. A 1:1 complex of actin and a 90,000 dalton protein has been purified from platelets. The modulation of actin assembly by this complex is currently being investigated.